1. Field of the Invention
The invention relates to compositions for the prevention and treatment of oxidant injury, particularly reperfusion injury, and to the method of use of such compositions and their components in mammals. Reperfusion injury has been observed to occur upon reperfusion following ischemia caused, for example, by blood clots, organic repair or transplant surgery.
2. Information Disclosure
A number of causes and mechanisms have been suggested for the damage that occurs to tissue after ischemia and reperfusion. While it is likely that a variety of causes and mechanisms contribute to the damage, a popular current theory that is supported by experimental evidence involves the generation of free radicals upon reperfusion. See J. L. Marx, "Oxygen Free Radicals Linked to Many Diseases," Science, Research News, 30 January 1987, pp. 529-531. The current consensus on the role of free radicals in reperfusion injury in the heart is discussed in a recent review article entitled "Free Radicals and Myocardial Ischemia and Reperfusion Injury," (Simpson and Lucchesi J. W. Lab. Med., July, 1987) which lists 146 references.
There are a number of reports in the literature of the use of superoxide dismutase (SOD) with or without catalase (CAT) for the prevention and treatment of reperfusion injury: Jolly et al. [Circulation Research 54, 277-285 (1984)] describe the beneficial effects of SOD plus CAT on infarct size in dogs. The SOD/CAT infusion is begun 15 minutes before a 90-minute occlusion and continues 15 minutes into reperfusion. Ambrosio et al. [Circulation 74, 1424-1433(1986)] describe the significant decrease in infarct size in the hearts of dogs treated with SOD at reperfusion after 90 minutes of proximal circumflex coronary artery occlusion. Similarly Ambrosio et al. [Circulation 75, 282-291(1987)] describe the significant improvement in isovolumic left ventricular developed pressure and phosphocreatine levels in Langendorff perfused rabbit hearts following 30 minutes of normothermic global ischemia when SOD was administered at reperfusion. The addition of CAT showed no benefit above that seen with SOD. Aoki et al. [Brit. J. Pharmacol. 95, 735-740(1988)] describe the significant attenuation of elevated post-perfusion pressure and protection against loss of myocardial creatine kinase activity in rats and perfused rat hearts upon administration of SOD after occlusion and after reperfusion. Werns et al. [Circulation Research 56, 895-898 (1985)] describe the beneficial effects of SOD but not CAT on infarct size in dogs after a 90-minute occlusion. European application 295826 discloses the use of SOD in open heart surgery to prevent reperfusion injury.
There have also been reports in the literature of the failure of SOD, with and without CAT, to effect any useful salvage of ischemic tissue: Uraizee et al. [Circulation 75, 1237-1248(1987)] reported that SOD before and during initial reperfusion after 40 minutes of circumflex coronary artery occlusion did not limit infarct size in dogs. Nejima et al. [Circulation 79, 143-153(1989)] reported that SOD and catalase did not improve regional myocardial dysfunction or infarct size in conscious dogs after 90-minute coronary artery occlusion. Klein et al. [Basic Res Cardiol. 83, 141-148 (1988)] disclose that recombinant human SOD administered two minutes before and for 45 minutes after initiation of reperfusion did not significantly reduce infarct size or diminish arrhythmia in pigs after 45-minute occlusion.
6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, also known as Trolox.RTM. C, has been reported to protect alcohol dehydrogenase from radiation damage [Gee et al. Brit. J. Radiol. 58, 251-256 (1985)] and to protect rat liver from the peroxidative damage caused by halobenzene poisoning [Casini et al. Am. J. Pathol. 118, 225-237 (1985)], although it did not protect isolated hepatocytes from herbicide poisoning [Sandy et al. Biochem. Pharmacol. 35, 3095-3101 (1986)]. Trolox.RTM. C was also reported to protect isolated perfused rabbit lungs from the pulmonary arterial pressure reaction provoked by the perfusion with a calcium ionophore; the effect was attributed to inhibition of cyclooxygenase and lipoxygenase activity by antioxidants [Wolf et al. Ann. N. Y. Acad. Sci. 393, 392-410 (1982) and Klinische Wochenschrift 59, 463-465 (1981)].
Kato et al. [J. Med. Chem. 31, 793-798 (1988)] reported that several acylated 2,3-enediol congeners of ascorbic acid were effective, when given prophylactically, in inhibiting reperfusion injury in the rat LAD model of myocardial ischemia. They further reported that both Vitamin E and ascorbic acid were without effect in this model. Woodward and Zakaria [J. Molec. Cell. Cardiol. 17, 485-493 (1985)] reported that ascorbic acid introduced after restriction and before reperfusion, reduced the incidence and duration of ventricular fibrillation in an isolated perfused rat heart model of reperfusion-induced arrhythmia.
The prophylactic use of Vitamin E to reduce the amount of tissue damage upon reperfusion of ischemic tissue in the kidney [Takenaka et al. Transplantation 32, 137-141 (1981)], in the brain [Fujimoto et al. Surg. Neurol. 22, 449-454 (1984)], and in the liver [Marubayashi et al. op. cit.] has been described. In the heart, the prophylactic use of Vitamin E has been reported to be successful in some paradigms [Cavarocchi et al. J. Surg. Research 40, 519-527 (1986) and Sarrett et al. Cardiologia 31, 539-544 (1986); Biol. Abs. 84063823 (1987)] but not in others [Ferrari et al. Acta Vitaminol Enzymol 7 Suppl., 61-70 (1985)]. Massey and Burton [Am. J. Physiol. 256, H1192-H1199 (1989)] reported significant protection from reperfusion injury in isolated, perfused rat hearts when the rats had been treated with 5 mg/day of Vitamin E by continuous release for 14 days before the induction of ischemia. Klein et al [Am. Heart J. 118, 667-673 (1989)] administered 12 g of Vitamin E acetate by i.v. infusion during a period of six days before occlusion of the left anterior descending coronary artery of pigs. In a separate group of pigs they administered 12 g of Vitamin E acetate by intraarterial infusion after occlusion and before reperfusion. In both groups ascorbic acid (0.1 g/kg) was infused i.v. after occlusion and before reperfusion. They reported that prophylactic treatment (the first group) was highly effective in preventing reperfusion injury; acute treatment was effective but only achieved "borderline significance".
We have shown [Mickle et al. Ann. Thorac. Surg. 47 553-557 (1989) and U.S. Pat. No. 4,877,810] that Trolox.RTM., and Trolox.RTM. with ascorbic acid are effective agents for the prevention and treatment of reperfusion injury. We have now discovered that SOD, catalase, Trolox.RTM., and ascorbic acid are selectively effective in different cell types to prevent and treat oxidative injury of the type that results from ischemia and reperfusion. Thus, as shown below, ventricular fibroblasts are more resistant to free-radical injury than are ventricular myocytes, which are, in turn, more resistant than arterial endothelial cells. Myocardial fibroblasts and arterial endothelial cells are best protected by SOD and catalase, whereas myocytes are best protected by Trolox.RTM. and ascorbic acid. This finding may help to explain the discrepant results reported for the utility of SOD/CAT and of Vitamin E in slightly differing models with differing treatment protocols and differing ways of assessing injury, all of which are based on whole-organ preparations. The results of whole-organ experiments usually reflect the mean contributions of all cell types in the organ affected. Differential effects on individual cell types resulting from ischemia-reperfusion injury, differential salvage by the treatment and differential detection by the assessment procedure are submerged in the aggregate result.